1. Field of the Invention
The present invention relates to an utrafiltration process for prepurification of complex biological matrices, in particular fermentation medium, which comprise a desired peptide or protein.
2. Description of the Related Art
To isolate and purify a protein or peptide from fermentation medium, for example, by means of chromatographic methods, a prepurification must first be carried out. In many cases, this comprises desalination of the fermentation medium.
The need to isolate a protein or peptide from fermentation medium typically arises in the context of recombinant microorganisms transformed with suitable expression vectors. Desired proteins or peptides, for the present ultrafiltration process, are typically a recombinantly produced protein or peptide. Often, the desired protein or peptide may contain an overall charge due to a greater overall concentration of basic or acid amino acid residues. A preferred protein or peptide contain an overall positive or negative charge of 2 or larger. Also preferred is a protein or peptide with an overall charge of 4 or larger.
The utility of this process may be demonstrated by the isolation of the thrombin inhibitor hirudin, a single-chain protein with 65 amino acids, from the culture supernatant of the yeast strain Saccharomyces cerevisiae modified by genetic engineering.
The polypeptide hirudin, originally isolated from the leech Hirudo medicinalis, is a highly specific thrombin inhibitor with a broad therapeutic potential (F. Markward, Biomed. Biochim. Acta 44 (1985) 1007-1013). Hirudin is characterized by a high proportion of dicarboxylic acids. Isolation from native sources is not commercially practical in light of the amounts required for medical utility; such an amount can be prepared only by a genetic engineering route via transformed microorganisms. It has been found in this context that the yeast Saccharomyces cerevisiae is a suitable host organism for producing correctly folded and fully active hirudin (EP A1 168 342, EP A1 200 655). Secretion of the protein results in concentrations of up to a few hundred milligrams of hirudin per liter of culture filtrate. A higher yield of the protein may be achieved if nutrient medium, or fermentation medium, is further enriched with additional nutrients, such as, yeast extract, corn steep, peptone or meat paste. Such additional nutrients are often protein-like substances, or contain additional protein from which the desired protein must be separated during the purification process. Therefore, use of enriched fermentation medium, while increasing protein yield, complicates ultimate purification of the protein, as there is the additional problem of isolating hirudin from a high dilution in a mixture of protein-like concomitant substances.
Methods of desalination of fermentation mediums as prepurification in preparation for chromatograph stages have included conventional methods, such as, extraction or precipitation, and hydrophobic adsorptions/desorptions, for example, on non-polar polymer materials (also HIC), (Atkinson, F. Mavituna; Biochemical Engineering and Biotechnology Handbook, Chapter 16 "Downstream Processing" and Chapter 17 "Product Recovery Processes and Unit Operations", Second Edition, Stockton Press 1991, New York, U.S.A; Brocklebank, M. Kalyanpur: "Primary Separation", Chapter 4 in G. Schmidt-Kastner et al., editors: "Recovery of Bioproduct", European Federation of Biotechnology, Study Report of Working Party on Downstream Processing, 1993; Muller and W. Brummer: "Die Chromatographie, eine zentrale Methode in der biotechnischen Aufarbeitung" [Chromatography, a central method in biotechnical workup], Chem. Ing.-Tech. 62 (1990) No. 5, pages 380-390). These processes usually involve large amounts of solvents or salts, compared with the amount of product to be prepared, which may result in additional costs or increased technical complexity for recovery and/or disposal of the solvents or salts. Furthermore, the spent adsorption resins are additional waste products.
In contrast, an object of the present invention is to provide, as prepurification process of peptide-containing fermentation medium, a membrane ultrafiltration process for the fermentation medium to a degree required for the subsequent purification steps, with high retention of the peptide or protein. The present process is particularly useful for desalination and concentration of the desired peptide.
Ultrafiltration processes for the prepurification of fermentation medium in preparation for chromatography stages have not been employed to date on a large industrial scale on these early process stages--apart from for the purpose of removal of cells (T .J. O'Sullivan et al. Chem. Eng. Prog. 80 (1), 68-75 (1984); A. Erikson, Desalination, 53 (1985), 259-263). One reason for this is that the membranes are often blocked by the by-products and concomitant substances, which may be diverse in nature, and therefore lead to slow permeate flow rates, which for production purposes are unsuitably low. Additional problems also result in the cleaning or regeneration of the membranes (Winzeler: "Membran-Filtration mit hoher Trennleistung und minimalem Energiebedarf" [Membrane filtration with a high separation efficiency and minimal energy requirement], Chimia 44 (1990) 288-291). The present process particularly applies to preparation of a protein of low molecular weight (M&lt;50,000 Dalton) and peptides, which supposedly require ultrafiltration membranes with very low molecular weight cut-off.
Processes are known where only at later process stages, after prepurification has already taken place, for example between successive chromatography stages, are ultrafiltrations performed, for example for desalination and concentration.
Membrane ultrafiltration processes are also used for separation of protein of different size, for removal of pyrogens or for isolation of biocatalysts (T. J. O'Sullivan et al., Chem. Eng. Prog. 80(1), 68-75 (1984); E. Flaschel et al., Adv. in Biochem. Engineering/Biotechnologie Volume 26, "Downstream Processing", pages 73-142, N.Y. 1983; Editor: D. J. Bell). EP 610 729 A1 discloses a process for the purification of protein or peptide by ultrafiltration, in which the nominal molecular weight cut-off of the ultrafiltration membrane is greater than the molecular weight of the protein to be purified, where the protein passes through the membrane.
Insulin undergoes reversible agglomeration, where higher aggregates, particularly insulin hexamers, under special conditions, dissociate. DD 225 943 A1 discloses an ultrafiltration process for the purification of insulin aggregates under special conditions favoring aggregation of insulin molecules. DD 225 943 A1 reference discloses a membrane with a molecular cut-off nearly equal to or smaller than the molecular weight of insulin aggromerates and insulin hexamers, but still larger than the molecular weight of single insulin molecule.
The processes mentioned are all based on the principle of employing, for filtration of a peptide or a protein, a membrane of molecular weight cut-off in or below the molecular weight range of the peptide or protein to be retained.